A Functional Proteomic Method for the Enrichment of Peripheral

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A Functional Proteomic Method for the Enrichment of Peripheral Membrane Proteins Reveals the Collagen Binding Protein Hsp47 Is Exposed on the Surface of Activated Human Platelets William J. Kaiser, Lisa-Marie Holbrook, Katherine L. Tucker, Ronald G. Stanley, and Jonathan M. Gibbins* Institute for Cardiovascular and Metabolic Research, School of Biological Sciences, Hopkins Building, The University of Reading, Whiteknights, Reading, United Kingdom RG6 6UB Received January 12, 2009

Platelets are small blood cells vital for hemostasis. Following vascular damage, platelets adhere to collagens and activate, forming a thrombus that plugs the wound and prevents blood loss. Stimulation of the platelet collagen receptor glycoprotein VI (GPVI) allows recruitment of proteins to receptorproximal signaling complexes on the inner-leaflet of the plasma membrane. These proteins are often present at low concentrations; therefore, signaling-complex characterization using mass spectrometry is limited due to high sample complexity. We describe a method that facilitates detection of signaling proteins concentrated on membranes. Peripheral membrane proteins (reversibly associated with membranes) were eluted from human platelets with alkaline sodium carbonate. Liquid-phase isoelectric focusing and gel electrophoresis were used to identify proteins that changed in levels on membranes from GPVI-stimulated platelets. Immunoblot analysis verified protein recruitment to platelet membranes and subsequent protein phosphorylation was preserved. Hsp47, a collagen binding protein, was among the proteins identified and found to be exposed on the surface of GPVI-activated platelets. Inhibition of Hsp47 abolished platelet aggregation in response to collagen, while only partially reducing aggregation in response to other platelet agonists. We propose that Hsp47 may therefore play a role in hemostasis and thrombosis. Keywords: Platelet • GPVI • Peripheral • Protein • Proteomic • Hsp47 • Collagen • Phosphorylation • Signaling • Membrane

Introduction Platelets are small, anucleate blood cells that are vital for hemostasis. Following adhesion to extracellular matrix proteins exposed at sites of blood vessel injury, platelets secrete a range of molecules that act in an autocrine or paracrine manner, enabling the recruitment, adhesion and activation of approaching platelets. Through platelet-platelet adhesion, a thrombus, or hemostatic plug is formed to stem the loss of blood.1 Platelet adhesion and subsequent thrombus formation is dependent on activation of cell surface receptors. The signaling cascades initiated through these receptors regulate and fine-tune the platelet response to vascular injury and control the extent of thrombus formation. Platelets may be activated, however, by vascular damage due to diseases such as atherosclerosis. This is a trigger for thrombosis, the formation of occlusive blood clots within an artery, resulting in hypoxia in downstream tissues. Thrombosis is the principal cause of heart attacks and strokes; therefore, understanding signaling mechanisms that regulate platelet functions is im* To whom correspondence should be addressed. Jonathan. M. Gibbins, E-mail: [email protected]. Tel: +44 (0) 118 3787082. Fax: +44 (0) 118 3787045. 10.1021/pr900027j CCC: $40.75

 2009 American Chemical Society

portant for the development of therapies to prevent or treat thrombosis.2 Collagens are the principal extracellular matrix proteins exposed at sites of vessel injury and platelets adhere to them via several receptors.3,4 The glycoprotein Ib-V-IX receptor complex binds to collagens weakly via von Willebrand factor and tethers the platelets temporarily, slowing them down, until stronger adhesion is achieved through other collagen receptors, such as integrin R2β1 and glycoprotein VI (GPVI). GPVI is essential for platelet activation and thrombus formation on exposure of platelets to collagens and initiates the predominant signaling cascade following platelet adhesion. Substantial progress has been made in characterizing GPVI signaling events in platelets, although identification of the proteins involved is incomplete, particularly those involved in later signaling events. Much of the current knowledge has been obtained through experiments involving co-immunoprecipitation (in order to find interacting partners of receptors and receptor-proximal signaling protein complexes), studies of receptors, adapter proteins, tyrosine kinases and phosphatases, and studying similarities with signaling pathways in immune cells have all been used to unravel GPVI signaling.3–5 Proteomic techniques involving mass spectrometry have impacted strongly on research toward the identification of platelet signaling proteins,6 and platelets Journal of Proteome Research 2009, 8, 2903–2914 2903 Published on Web 04/02/2009

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hold an advantage as a simple system for proteomic analysis due to lack of a nucleus. Existing proteomic techniques for the characterization of signaling pathways are frequently hindered by the low concentrations of these proteins relative to other more abundant proteins (such as structural proteins) in the cell. Furthermore, analysis of protein complex formation using techniques such as immunoprecipitation, requires prior knowledge of relevant complex components and the availability of suitable antibodies for protein isolation with complexes remaining intact.7 Sample fractionation presents a major challenge for functional proteomic studies and the frequent incorporation of multiple steps complicate comparative analysis due to variable sample recovery. Maintaining post-translational modifications and protein-protein interactions is necessary if using these as a handle to enrich signaling proteins for comparative analysis and these may be lost due to the prolonged manipulations during fractionation. Our aim was to develop a method that allows the enrichment of signaling proteins without extensive manipulations and without the loss of post-translational modifications, in order to allow samples to be compared for functional studies. A common feature in all cells is the recruitment of signaling proteins to the inner-face of the plasma membrane upon cell surface receptor activation.8–10 For instance, when platelets are activated through GPVI, the FcRγ-chain (noncovalently associated with the cytoplasmic tail of GPVI) becomes tyrosine phosphorylated within the immuno-receptor tyrosine-based activatory motif (ITAM)11 and the tyrosine kinase Syk is recruited and binds via its Src-homology 2 (SH2) domains.12 Syk phosphorylates LAT (a transmembrane adapter protein), which forms a hub for the assembly of a complex of signaling proteins, such as phosphoinositide-3-kinase (PI3K) and phospholipase Cγ2 (PLCγ2), that collectively contribute to the activation of platelet function.13,14 At the membrane, the activity of these proteins is controlled with precision and signaling proteins, present in low concentrations throughout the cell, become concentrated in close proximity to their substrates. Isolating protein complexes from membranes could therefore provide an enriched fraction of signaling proteins, facilitating their identification with mass spectrometry. Receptor-proximal signaling proteins behave as peripheral membrane proteins (PMPs), a class of proteins that interact with membranes only temporarily via noncovalent interactions with proteins embedded in the lipid bilayer (integral membrane proteins) or the membrane lipids themselves.10 PMPs may associate with either the internal or external surfaces of membranes. A number of proteins secreted from platelets, such as PDI and ERp5, associate with the platelet surface and these are therefore external PMPs.15 These interactions are readily disrupted with detergents, chelating agents and buffers of high ionic strength. Therefore, in order to enrich for PMPs on membranes, it is necessary to avoid the use of detergents and high salt buffers. An aim of this study was to identify platelet proteins recruited to membrane signaling complexes upon GPVI activation. We have developed a proteomic-compatible method for isolating PMPs as a distinct fraction from other proteins, such as cytosolic or integral membrane proteins. This method allows PMPs to be isolated without bias, unlike detergent-based coimmunoprecipitations that would only permit the recovery of tightly interacting proteins bound to the antibodies of choice. 2904

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Figure 1. Verifying elution of PMPs from platelet membranes. Samples from each stage of PMP preparation were probed for proteins of known subcellular localization by Western blot (L ) platelet lysate). Integrin β3, an integral membrane protein, was only detected in the pellets obtained after the first (P1) and second (P2) high-speed centrifugation (containing platelet membranes). GAPDH, a cytosolic protein, was largely detected in the supernatant after the first high-speed centrifugation (S1). 14-33ζ, a peripheral membrane protein, was detected in all samples and was eluted with sodium carbonate (S2). Representative of 4 separate experiments.

This functional proteomic approach was used to compare PMPs from inactive (resting) platelets with PMPs from platelets stimulated with collagen related peptide (CRP; a specific GPVI agonist16). Proteins that translocated to and from membranes upon activation through GPVI could therefore be identified. Levels of protein phosphorylation were maintained throughout the enrichment, enabling phosphorylation-dependent protein interactions to be maintained. The isolation of PMPs, combined with mass spectrometry, was found to be an effective approach for the discovery of lowabundance signaling proteins. To validate this methodology, platelet PMPs were analyzed by mass spectrometry. Heat-shock protein 47 (Hsp47), a collagen binding chaperone protein, was detected from the PMP fraction from GPVI activated platelets. Flow cytometry revealed Hsp47 was present on the platelet surface, the levels of which increased following platelet stimulation with CRP. The effects of a selective inhibitor of Hsp47 implicate this molecule in the responses of platelets to collagen.

Materials and Methods Reagents used were of analytical grade and obtained (unless stated) from either Sigma (Poole, U.K.) or Fisher Scientific (Leicestershire, U.K.). Anti-p38 total and phosphospecific antibodies were obtained from Cell Signaling Technology (Heartfordshire, U.K.), anti-14-3-3ζ, β3 (Santa Cruz, CA), p85 (Upstate, MA), GAPDH (Abcam, Cambridge, U.K.), and anti-phosphotyrosine (Upstate). Hsp47 inhibitor was obtained from Calbiochem (Darmstadt, Germany). CRP was purchased from Prof. Richard Farndale (University of Cambridge). Platelet Preparation and Stimulation. Blood was obtained from healthy, drug-free volunteers as approved by the local research ethics committee. Blood was drawn into 50 mL syringes containing 3 mL of 4% (w/v) sodium citrate and 7.5 mL of acid citrate/dextrose (10% w/v) added prior to centrifugation at 100g for 20 min at room temperature. The top 75% of platelet rich plasma was harvested and pooled into a 50 mL tube, with prostacyclin (PGI2) at 50 ng/mL, and centrifuged for 10 min at 1400g to pellet platelets. Platelets were resuspended in 25 mL of modified Tyrodes-HEPES buffer (134 mM NaCl, 2.9 mM KCl, 0.34 mM Na2HPO4, 12 mM NaHCO3, 1 mM MgCl2, 20 mM HEPES and 5 mM glucose, pH 7.3), 3 mL of ACD and

Enrichment of Platelet Peripheral Membrane Proteins

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Figure 2. Preservation of tyrosine phosphorylation throughout sample fractionation and the recruitment of GPVI signaling pathway components to the PMP fraction. Samples from each stage of the procedure (derived from resting and GPVI-activated platelets) were probed with an anti-phosphotyrosine antibody by Western blot (a). A longer exposure of (a) showed a large proportion of the tyrosine phosphorylated proteins present on the platelet membranes (P1) were eluted with sodium carbonate (S2) (b). The regulatory subunit of PI3K, p85, increased in levels on the membranes of activated platelets (P1) and was eluted with sodium carbonate (S2) (c, top). A reprobe for LAT demonstrated equal sample loading (c, bottom). Figures are representative of 3 separate experiments.

50 ng/mL PGI2. Platelets were counted on a Z2 Coulter particle counter and centrifuged again at 1400g, before resuspension in modified Tyrodes-HEPES buffer to a density of 2 × 109 platelets/mL. For optical aggregometry, platelets were resuspended to a density of 4 × 108 platelets/mL. Platelet preparations typically contained fewer than 1 contaminating erythrocyte or leukocyte per 6500 platelets. To restrict the platelet signaling response downstream of GPVI, inhibitors of secondary signaling were included. Indomethacin

was added (10 µM) to inhibit thromboxane production and platelets were incubated for 1 h at 30 °C prior to use. Apyrase (2 U/mL) was added to reduce signaling through P2Y1 and P2Y12 receptors via secreted ADP, and 2.92 mL of platelets (5.84 × 109 platelets) was transferred to each of four 15 mL tubes. Samples were incubated for 2 min with Eptifibatide (4 µM final) at 37 °C to prevent fibrinogen-mediated aggregation and signaling through RIIbβ3. Platelets were activated with 10 µg/mL collagen related peptide (CRP), a specific ligand for GPVI or carrier alone (TyrodesJournal of Proteome Research • Vol. 8, No. 6, 2009 2905

research articles HEPES buffer). Tubes were mixed by inversion and incubated for 2 min at 37 °C. Platelet stimulation was terminated by snap freezing in liquid nitrogen. For Hsp47 inhibitor experiments, platelets were incubated at 37 °C for 2 min with either Hsp47 inhibitor (Calbiochem, Darmstadt, Germany) or vehicle control (0.12% DMSO), prior to optical aggregometry (Chrono-log, Havertown, PA). Platelets were stimulated with either collagen (1 µg/mL), CRP (0.5 µg/ mL), convulxin (60 ng/mL), thrombin (0.1 U/mL) or vehicle control (Tyrodes-HEPES), while stirring, for 2 min at 37 °C, and aggregation trace was recorded. Preparation of Cleared Platelet Lysates. Ice-cold inhibitor stock (2.5 µg/mL Pepstatin A, 1 µg/mL Leupeptin, 1 µg/mL Aprotinin, 10 mM Na3VO4 and 5 µM Staurosporine in modified Tyrodes-HEPES buffer) was added to each tube (168 µL) of frozen platelets (stored at -80 °C), and samples were thawed completely in a 37 °C water bath. The samples were snapfrozen again and the freeze-thawing was repeated a further two times. Samples were then sonicated for 3 min in a Transsonic T310 water bath (Camlab) containing iced water. Finally, samples were centrifuged at 4500g for 5 min at 4 °C and 2.8 mL of supernatant was collected. Pairs of resting samples and CRP-stimulated samples were combined (5.6 mL of each). Preparation of PMPs from Cleared Platelet Lysates. Inhibitor stock (168 µL) was added to each 5.6 mL sample of cleared platelet lysate and transferred to centrifuge tubes on ice. Samples were centrifuged at 47 800g for 1 h at 4 °C in a Beckman SS-34 rotor. Supernatant was removed (the cytosolic fraction) and the pellet (total membranes) was washed gently with 1 mL of ice-cold tyrodes, followed by resuspension in 2 mL of ice-cold 100 mM Na2CO3 at pH 11.5. Samples were incubated on ice for 1 h with the addition of 84 µL of inhibitor stock, followed by centrifugation again at 47 800g for 1 h at 4 °C. The supernatant was collected, containing PMPs and the pellet washed with 1 mL of modified Tyrodes-HEPES, followed by resuspension in 2 mL of NP40 buffer (1% NP40 (v/v), 150 mM NaCl, 10 mM Tris and 5 mM EDTA), representing the integral membrane protein fraction. Immunoblot Analysis. At each stage of the fractionation, 100 µL of sample was taken and immediately mixed with 100 µL of reducing Laemmli sample buffer in preparation for SDS-PAGE. Samples were separated on large format 10-18% SDS polyacrylamide gradient gels using a BioRad Protean II xi unit and transferred to BioRad Immun-Blot polyvinyledene difluoride (PVDF) using a BioRad Trans-Blot semidry blotter. PVDF membranes were blocked with 5% BSA (w/v) in Tris-buffered saline-Tween (TBST; 20 mM Tris, 137 mM NaCl, 0.1% Tween 20 (v/v), pH 7.6) and washes were performed using TBST. Antibodies were diluted 1/1000 (primary) or 1/4000 (secondary) in 2% BSA (w/v) in TBST. Proteins were detected using Pierce ECL substrate and exposure to X-ray film. Liquid-Phase Isoelectric Focusing and Gel Analysis. Whole peripheral membrane fractions (2 mL) were dialyzed in TubeO-Dialyzers (4 kDa cutoff, from G-Biosciences) against 2 × 500 mL 0.01 M acetic acid at 4 °C. Following dialysis, sample volumes were adjusted to 3 mL with nanopure water and the focusing buffer components added directly to the sample (410 mg urea, 76 mg thiourea, 5 mg CHAPS, 5 mg DTT, 50 µL glycerol and 50 µL Triton-X 100). Samples were rotated at room temperature for 15 min and 80 µL of ampholytes (BioRad, 40% (w/v), pH 3-10) was added immediately prior to focusing. Samples were injected into the focusing chambers of a BioRad 2906

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Kaiser et al. MicroRotofor (BioRad, Hemel Hampstead, U.K.) and separated for 2 h on a step up cycle (150 V for 15 min, 200 V for 15 min, and 300 V for 1.5 h, all limited at 2 W and 20 mA). Following separation into 10 fractions, 1 mL of 100% acetone at -20 °C was added and samples were precipitated overnight at -20 °C. Tubes were centrifuged at 10 000g and the pellets resuspended in a sonicating water bath for 5 min, in 100 µL of reducing Laemmli sample buffer/0.05 M NaOH. Samples were heated at 90 °C for 10 min and loaded onto gradient gels as for Western blots. Gels were silver-stained as described previously.17 Trypsin Digestion and Liquid Chromatography (LC), followed by Fourier-Transform Ion Cyclotron Resonance (FT-ICR) Mass Spectrometry (MS/MS). In-solution trypsin digestion, in-gel digestion and subsequent FT-ICR analysis were performed as a service by the Genomics Suite at The University of Birmingham (U.K.). Trypsin digestion was performed with an automated system. MS/MS was conducted using a Thermo Finnigan LTQ-FT, comprising a 7 T FT-ICR with a front-end linear ion trap. LC runs were 2 h using a 5-60% gradient of 0.1% (v/v) formic acid in water and 0.1% (v/v) formic acid in acetonitrile. MS/MS data were analyzed using a TurboSEQUEST search algorithm against a human-primate database. For the whole in-solution digest, proteins were considered to be reliably identified with a probability (P) value of 25

23

250

15

15

46

75 >37

31 15

20

14

38

28.1 17.3 40.1 30.1 47.2 15.1 20.4 37.1 31.3 13.5 12.9 30.9 21 31.3 13.7 7.7 45.1 18.7 15.9 7.8 12 26.5 60.1 26.8 38.2 29 32.9 23.6 39.7 15.4 27.8 41 10.7 6.7 16.1 33.5 12.7 49.7

function HK Sig C Sig C C Sig HK HK HK C HK C HK HK Sig C S HK Sig Sig Contaminant? HK HK HK Sig HK HK Sig C Sig HK Sig S Sig HK Sig C

a Following trypsin digestion, peptides were identified by LC MS/MS. The column headed ‘peptides’ refers to the number of peptides with P < 10-5. Key: Transmembrane protein (TM), cell signalling protein (Sig), secreted (S), house keeping/metabolic (HK) and structural/regulatory protein of cell skeleton (C). Peptide sequences are listed in Supporting Information.

of the proteins identified were known to be associated with the membranes of activated platelets, including ILK27,28 and 14-3-3ζ.19,20 The p38 isoform of MAPK was identified from one of these bands and Western blot analysis showed it was present in the sodium carbonate eluted fraction. However, levels of total p38 only increased in the activated fractions from some individuals. It is possible that p38 may have changed in levels, in individual fractions separated by SDS-PAGE, due to a shift in isoelecric point upon phosphorylation. Platelets were obtained from human blood, so donor variation may have been responsible for these inconsistencies (variability in response to collagen is well-documented). Nearly all the proteins identified from the gel slices, and from the total sodium carbonate eluted protein digest, have been shown to associate reversibly with membranes or with proteins embedded in membranes in platelets and other cell types. Only two proteins identified in this way were integral/transmembrane proteins, demonstrating the high degree of reliability of the technique for isolating only PMPs. Heat-shock protein 47 (Hsp47) was identified from a band that increased in staining intensity in the stimulated sample, and was previously unconfirmed in platelets. Hsp47 was first identified as a collagen-binding chaperone protein in cells that

secrete collagen.29 More recent work has demonstrated it to be exposed on the surface of tumor cells via an interaction with tetraspanins (CD9), and it has been proposed that it may be a type of collagen receptor.23 PMPs may associate with either the inner leaflet of membranes or the external surface. The presence of Hsp47 on the surface of platelets was therefore investigated using flow cytometry. The levels of Hsp47 were found to increase on the surface of platelets activated with CRP. This is consistent with Hsp47 either being secreted in a soluble form following activation and then binding back to platelet surface, or complexing with another membrane protein that is up-regulated on the surface. An inhibitor of Hsp47 greatly attenuated platelet aggregation induced by collagen fibrils, preventing aggregation at 10 µM and only allowing shape change at 5 µM. The inhibitor was not effective at 10 µM at stopping aggregation in response to CRP, or at 20 µM with convulxin, although a reduction in aggregation was observed. The inhibitor did not reduce aggregation in response to thrombin. All agonists were used at concentrations that produced a similar level of aggregation with agonist alone. Hsp47 can bind triple-helical collagen;30 the structure of collagens platelets would be exposed to at sites of vessel injury. Convulxin, however, is a snake venom protein Journal of Proteome Research • Vol. 8, No. 6, 2009 2913

research articles that has no similarity to collagen, so inhibition of convulxinstimulated platelet aggregation by the Hsp47 inhibitor suggests that the mechanism that Hsp47 may utilize in platelets is not straightforward and it may be more than just a collagen binding protein involved with adhesion. Further studies are required to investigate whether Hsp47 may be involved with platelet adhesion to collagen fibrils and behave as a type of collagen receptor. The enrichment of PMPs from isolated platelet membranes was found to produce a fraction that is enriched for signaling proteins. Moreover, the preservation of protein phosphorylation allowed samples from resting and stimulated platelets to be compared to identify proteins that change in levels within the fraction or post-translational modifications, such as phosphorylation. With this method, the collagen chaperone protein Hsp47 was identified as a protein that may contribute to platelet function in the initial stages of platelet interaction with collagen. This method allows the possibility of not only identifying novel cellular proteins, but also attributing them to specific signaling pathways, making this procedure useful for comparative or functional proteomics in a range of cell systems.

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Acknowledgment. This research was supported by

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grants from the Medical Research Council (G0400883), British Heart Foundation (FS/07/018) and the Wellcome Trust (082338/7/07/7 and 072498/7/03/7).

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Supporting Information Available: Peptide sequences for proteins identified in Tables 1 and 2 (Excel file: peptide sequences). Supplementary Figure 1, flow cytometry showing that Hsp47 immunoreactivity also increased on the surface of thrombin-stimulated platelets. This material is available free of charge via the Internet at http://pubs.acs.org.

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